In the past, a basic assumption of plant biologists and biochemists is that the internal tissues of plants are sterile. Any microbial activity in cell cultures or tissue extracts as attributed to contamination as opposed to indigenous bacteria. Rarely is it considered that the contaminant might have been associated with the plant in nature in a biologically meaningful way. A more pernicious assumption is that if there is no apparent microbial contamination in a culture or extract, then the enzymatic activities present in the plant must be plant activities themselves. That is, all activities related to the plants, such as enzymatic activities, are directly related to indigenous plant enzymes. Applicant has been forced to reconsider these assumptions with the findings that an enzymatic activity in leaf tissue and cell cultures of soybean is of bacterial origin.
Upon applicant's discovery of the commensal bacteria, it still did not remain clear whether one could distinguish bacterial from plant activity in whole tissue assays and whether the bacteria made any contribution to plant metabolism or to what extent the plant influences the metabolism of the bacteria.
Previously, plant production has been altered in various ways. For example, genetic engineers have attempted to modify the genome of plants directly to alter agronomic performance. Of course, classic methods of grafting and cross pollinating plants have been used.
Applicant herein discloses a discovery of commensal bacteria which have a significant contribution to the enzyme activity of the host plant. Further, applicant has further demonstrated that plants can be cured, in whole or in part, of their bacterium and that activation of at least two bacterial enzymes is achieved by the plant's metabolic machinery. Thus, there is a close probability of coadaption and association between the bacterium and its plant host. Utilizing this relationship, applicant will increase the agronomic performance of the plant reintroducing genetically altered or engineered strains into cured plants thus endowing the host plant with improved agronomic performance.
There have been other reports in the literature that biochemical activities in plants may be due to naturally associated non-pathogenic bacteria (1,2,3). It seems that these are treated as exceptional examples of symbiosis and are not taken as indicative of a general biological phenomenon. There is little doubt that many of the activities of microbial commensals associated with plants are insignificant to the plant.
Pink-pigmented, facultative methylotrophs (PPFM's) were first described in connection with plants more than 20 years ago when it was demonstrated that cell cultures of the leafy liverwort Scapania are routinely associated with PPFM's (4). In the years since that time, PPFM's have been isolated for more than 60 species of plants, including both nonvascular and vascular plants, gymnosperms and angiosperms, dicots and monocots (5). These observations have lead to the speculation that the bacteria are universally associated with plants, but the exact nature of the relationships has remained obscure.
The bacteria are seed transmitted and have been seen by electron microscopy to occupy cavities in the cuticle in the leaf tissue (6). Basile et al. has shown that liverworts cultured in the absence of PPFM's require supplementary vitamin B-12 and that vitamin B-12 is produced by the bacteria (7). Although it has long been supposed that plants do not produce vitamin B-12, cobalamin-dependent enzyme activities are known to exist in plants (8,9). It has also recently been reported that plants produce free methanol which suggests that the availability of an exploitable resource for the PPFM is not accessible by other phylloplane bacteria (6).